Enzyme-free, Label-free Quantitative Microarray Technology: Fundamentals to Validation - SUMMARY Measuring immunospecific binding of target nucleic acids (NA) to single stranded DNA (ssDNA) probes immobilized on solid surface has revolutionized molecular biology analysis from basic science to medicine. However, microarray-based technologies including Next Generation Sequencing can detect mutations with high precision but have limited ability to quantify copy numbers. The gold standard for NA quantification, qPCR is limited to meet the current need to quantify small NA, such as miRNA at required sensitivity in attomolar (aM) range for applications such as liquid biopsy for diagnosis and prognosis. Label-free, enzyme-free electro- chemical approaches, particularly Electrochemical Beacon (EcB) is a promising technology to make microarray quantitative. However, the limit of quantification (LOQ) is only ~10 pM due to the complexity of the probe design which is a folded structure with complex conformation. In the proposed EcB study a simple binary probe is designed comprising of short reporter DNA (R) tethered to the electrode with longer probe (P) complimentary to the target (T) of interest. The R has a redox active dye, methylene blue (MB) tethered to the free-end. On P-T binding the released R brings MB in proximity to the electrode resulting in a signal. The research strategy is to design an R-P probe such that there is no signal from the binary probe and a signal is generated on each P-T binding, i.e., positive contrast. The background is well controlled to have low false positives and negatives. The goal is to use a special opto-electrochemical tool called SEED to read P-T binding to a microarray of binary probes on a monolith electrode by measuring local redox on 10 micron spots to obtain LOQ of 10 attomolars with dynamic range of over seven orders of magnitude to profile a mixture of ~25 miRNA sequences on a chip. The premise is based on previously published result showing SEED has the required sensitivity. The study will be organized in two specific aims: Specific Aim 1: Binary Probe design verification. The goal will be to, (a) create a “perfect switch” where before binding all (R-P) probes show no signal and, on each P-T binding the released R contributes to a redox signal. and (b) establish calibration curves to quantify mixtures of up to 25 synthetic ssRNA (or analogous ssDNA) targets with background of non-specific NAs. Specific Aim 2: Validation of binary probe design. The goal will be to quantify ~25 miRNA in cell line and exosomes suspended in its media. The cells will be cultured with/without UV exposure. The success will be a quantitative profile of ~25 miRNA sequences in biological sample with [c] ranging from ~10 aM to ~1 nM. On success, collaborations will be initiated to translate the application to 3D culture (organoid/spheroid), stem cell differentiation and liquid biopsy applications.
